title: Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts.
abstract: We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the ¹⁵N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D ¹H-¹⁵N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.
authors.affiliation: In-Cell NMR Laboratory, Department of NMR-supported Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin), Berlin, Germany.
authors.forename: Francois-Xavier
authors.initials: FX
authors.lastname: Theillet
authors.forename: Honor May
authors.initials: HM
authors.lastname: Rose
authors.forename: Stamatios
authors.initials: S
authors.lastname: Liokatis
authors.forename: Andres
authors.initials: A
authors.lastname: Binolfi
authors.forename: Rossukon
authors.initials: R
authors.lastname: Thongwichian
authors.forename: Marchel
authors.initials: M
authors.lastname: Stuiver
authors.forename: Philipp
authors.initials: P
authors.lastname: Selenko
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000032:analysis*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D012694:Serine / Q000032:analysis / Q000378:metabolism*; D013912:Threonine / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D011506:Proteins; D016159:Tumor Suppressor Protein p53; D013912:Threonine; D012694:Serine; D011494:Protein Kinases